When p33ING1 (ING1) was shown to physically and functionally interact with p53, we hypothesized that p33ING1 was the proband member of a family of genes encoding p53 co-transcription factors, and thus, initiated a search for ING1-related genes. By using methodologies already established in the LHC, we have identified four related genes (p47ING3, p29ING4 and p28ING5; ING2-5). We are systematically investigating each of these members of the ING gene family. For example, we have previously reported that unlike ING1b, an alternatively spliced form of ING1, ING2, is induced by the DNA-damaging agents, etoposide or neocarzinostatin. ING1b or ING2 negatively regulate cell growth and survival in a p53-dependent manner through the induction of G1-S cell cycle checkpoints and apoptosis. ING2 strongly enhances the transcriptional transactivation activity of p53. Furthermore, ING2 expression increases the acetylation of p53 at lysine-382. These results have been recently confirmed and extended by others, i.e., ING2 is a nuclear phosphoinositide receptor. Taken together, ING2 is a DNA damage-inducible gene that negatively regulates cell proliferation through the activation of p53 by enhancing its acetylation. Our recent data from SiRNA knockdown of ING2 also indicates that ING2 can contribute to the induction of the scenescent phenotype. In collaboration with Karl Riabowol, we found that a subset of ING family members strongly repressed human alpha-fetoprotein (AFP) promoter activity, but stimulated the p21(WAF1) promoter in parallel experiments in the same cell type, similar to the effects of p53. The p47(ING1a) isoform also repressed AFP promoter activity, but in contrast to other ING isoforms, it repressed the p21(WAF1) promoter. p47(ING3) upregulated p21(WAF1) promoter activity, but it did not have any effect on the AFP promoter. ING1b and ING2 also repressed the AFP promoter in Hep3B p53-null cell lines, and p53 coexpression enhanced this transcriptional repression. Suppression of AFP gene transcription by ING was strongly dependent on AT-motifs that bind to the hepatocyte nuclear factor 1 (HNF1) transcription factor. Indeed, electrophoretic mobility shift assays confirmed that HNF1 binds to AT-motifs, but we found, surprisingly, that the ING1 complexes binding to these AT-motifs were devoid of HNF1 protein. Both ING1 and p53 were able to suppress AFP transcription and cause p21 induction; hSIR2, a negative regulator of the p53 protein, showed the opposite effects on the AFP promoter and, like HDAC1, repressed p21 promoter activity. In addition, we found that p33(ING1b) physically interacts with hSIR2, reverses its ability to induce the AFP promoter, and induces acetylation of p53 residues at Lys(373) and/or Lys(382). These findings provide novel evidence that p33(ING1b) represses AFP transcription by at least two mechanisms, one of which includes p53. The first is by binding to the AT-motif and excluding HNF1 binding while possibly targeting HAT activity on promoter regions, and the second is by increasing the levels of active, acetylated p53 via binding and inhibiting the ability of hSIR2 to deacetylate p53 protein. We have discovered that ING3-5 also participate in the p53 response pathway to cellular stress. Our preliminary data indicate that different members of the ING family physically and functionally interact with multiprotein complexes of either histone deacetylases, e.g., SIR2, or histone acetyltransferase, e.g., p300, and coregulate p53-mediated transcription by chromatin remodeling.